1. Field of the Invention
The present invention relates to the field of drug development and, in particular, to a high volume screening process used to measure the effects of compounds on enzyme activity. The present invention is a method and device that eliminates the difficulties associated with the utilization of fluorescent methods in the presence of unknown compounds. More particularly, it is a fluorescent method that includes a synthetic particle for determination of neutral lipid transfer protein activity with provisions to correct for interference that would normally occur during the measurement of the activity, and is a device that carries out this method or test.
2. Description of the Prior Art
Neutral lipid transfer proteins include both cholesteryl ester transfer protein (CETP) and microsomal transfer protein (MTP). CETP is a protein that transfers cholesteryl ester (CE) from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) and very-low density lipoprotein (VLDL). CETP will also transfer triglyceride (TG) among lipoprotein particles. For example, when a sample of VLDL or LDL, 1 or 10 micrograms of protein, respectively, is mixed with a sample of HDL, at total HDL cholesteryl ester of 4EE-10 moles, incubated at thirty seven degrees centigrade with a source of CETP, such as, one microliter of human plasma, cholesteryl ester will be transferred from the HDL to the LDL or VLDL particles.
MTP is an intra-cellular protein believed to be associated with the endoplasmic reticulum (ER) of normal liver and intestinal cells. MTP is also believed to be responsible for the synthesis and secretion of very low density lipoprotein (VLDL). If 100 microliters of liver cell homogenate is added to 4EE-10 moles of HDL cholesteryl ester and 1 ug of VLDL protein at 37 degrees centigrade for 12 hours, transfer of neutral lipid among the lipoproteins will occur.
Typically, the measurement of the CETP or MTP activity requires the cholesteryl ester associated with the HDL be provided with some type of label for monitoring movement of the HDL cholesteryl ester to the LDL and VLDL components after incubation. The activity measurement techniques also usually require a final separation step after incubation so that either accumulation of HDL cholesteryl ester in LDL or VLDL may be quantified, or loss of HDL cholesteryl ester from HDL may be quantified. The HDL particle represents a donor of CE, and the VLDL or LDL represent acceptors of CE.
There are several known techniques to measure cholesteryl ester transfer protein (CETP) activity. For example, an article entitled: Effect of Very Low-Density Lipoproteins on Lipid Transfer in Incubated Serum, by A. V. Nichols and L. Smith, J. Lipid Research, vol. 6, pp. 206-210 (1965), measures the activity of CETP by determination of cholesteryl ester (CE) mass transfer. The determination of CE mass transfer from high density lipoprotein to very-low density lipoprotein (VLDL) and low density lipoprotein (LDL) requires the re-isolation of VLDL and LDL after incubation with HDL and the CETP source in order to determine the cholesteryl ester mass transfer.
The VLDL/LDL re-isolation from the incubation mixture is a technique that includes ultra-centrifugation of the incubation mixture for many hours so that the VLDL and LDL components are floated upwards through a density gradient as the HDL component of the incubation mixture sinks to the bottom of the centrifuge tube. Further processing of the sample requires a method of determining the amount or mass of cholesteryl ester associated with the re-isolated VLDL or LDL and equating a change in mass to CETP facilitated transfer. Later variations of this method of activity measurement have simplified mass determination by utilizing HDL that has a radioactive label associated with the CE.
While not stated in this article, the determination of CETP activity through tritium (3H) labeled cholesteryl ester (3H-CE) still requires the time consuming step of VLDL/LDL component re-isolation, or separation of VLDL or LDL from the 3H-CE containing HDL before the disintegrations per minute (DPM) of 3H-CE transferred can be determined.
The present method does not require the separation of any components of the incubation mixture, nor does the present method use radioactive isotopes.
An article entitled: Cholesteryl Ester Exchange Protein in Human Plasma Isolation and Characterization by N. M. Pattnaik, A. Montes, L. B. Hughes and D. B. Zilversmit, Biochemica et Biophysica Acta 530, pp. 428-438 (1978), discloses a method of activity measurement of CETP that also utilizes radioactive CE in HDL. This method is an improvement over the above method by simplifying the incubation mixture components separation or the re-isolation technique discussed above. In the cited article, separation of the LDL component from the HDL component is accomplished by precipitation of the LDL component of the incubation mixture. The LDL precipitate is pelleted by a relatively short slow-speed centrifugation and the remaining HDL supernatant is counted. The loss of radioactivity from the HDL component is attributed to 3H-CE transferred to the LDL pellet. This method requires the use of radioactive isotopes and it is believed both prior art publications yield poor sensitivity and accuracy, characteristic of methods that require a high incident of sample manipulation.
An article titled: Fluorescent Determination of Cholesteryl Ester Transfer Protein (CETP) Activity in Plasma by N. Dousset, L. Douste-Blazy in Clinical Chemistry, vol. 38, No. 2, p. 306 (1982), is an improvement over previous methods of activity measurement since it discloses a technique that does not require radioactive components. In this method, transfer activity of the CETP is determined by the measurement of transfer of a fluorescent labeled CE. In this reference, the cholesteryl ester molecule utilized as the CETP substrate for transfer has been covalently bound to a fluorescent molecule derived from pyrene. The pyrene labeled cholesteryl ester (PY-CE) is recognized by the CETP and the PY-CE may be detected by a fluorimeter. The accumulation of the PY-CE in the LDL fraction is, however, only able to be determined after the separation of the LDL acceptor from the HDL donor.
An article entitled: Enhancement of The Human Plasma Lipid Transfer Protein Reaction by Apolipoproteins by T. G. Milner, K. W. S. Ko, T. Ohnishi, and S. Yokoyama in Biochimica Biophysica Acta 1082, pp. 71-78 (1991), discloses a method for determining the activity of CETP also utilizing a pyrene labeled CE (PY-CE). This method does not require separation or re-isolation of substrates, but uses the measurement of both monomer and excimer fluorescent emission from the pyrene label to determine a ratio thereof. The cited article is improving upon certain aspects of the previous method. However, the method is based upon excimer to monomer ratio to determine accumulation of PY-CE in the acceptor and does not account for lipoprotein core viscosity changes affecting the excimer to monomer ratio. Pyrene labels have been used extensively in physical biochemistry to study particle core viscosity. This cited method is a technique that results in problems with accuracy as noted in this article. In addition, the method is inconvenient due to oxygen quenching of excimer emission and requires the constant gassing of samples with nitrogen.
An article entitled: Use of Fluorescent Cholesteryl Ester Microemulsions in Cholesteryl Ester Transfer Protein Assays by Charles L. Bisgaier, Laura Minton, Arnold D. Essenberg, Andrew White, and Reynold Homen published in the Journal of Lipid Research, Volume 34, 1993 discloses a method that utilizes a self quenching florescent labeled cholesteryl ester core. The reference cited, although an improvement, does not address the problems associated with emulsion instability and, as pointed out by the authors, spectral interference due to the presence of colored compounds.
An article entitled: Protein Disulfide is a Component of the Microsomal Triglyceride Transfer Protein Complex by Wetterau, J. R.; Combs, K. A.; Spinner, S. N. and Joiner, B. J. discloses a method of measuring MTP activity utilizing radioactive isotopes.
The priority application, noted above, will not be applicable in instances where the addition of colored compounds accompany neutral lipid transfer protein activity measurements.
The present method is readily usable for the purpose of performing simple, rapid and accurate tests to determine activity of CETP or MTP in the presence of compounds that may otherwise prevent accurate measurement. This present method accomplishes this without the use of radioactive substrates, utilizing a stable emulsion substrate without requiring separation of donor and acceptor particles to quantify activity measurements. Further, it yields a real time activity and the reagent substrate emission is not subject to quenching by oxygen. Still further, the fluorescent emission spectra monitoring the enzyme activity is correctable in the event of spectral interference.